JPH1149793A - Antitumor substance originated from vegetable - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new substance originated from vegetables, having a specific mol.wt., capable of being extracted with organic solvents, capable of being developed and separated with high performance tin layer chromatography and having a selective proliferation-inhibiting activity against tumor cells, and useful for antitumor agents, etc. SOLUTION: This new antitumor substance is originated from a vegetable such as Raphanus sativus L. var., Allium sativum L., or Zingiber officienale, has a mol.wt. of <=3500 measured by a dialysis membrane, can be extracted with organic solvents, is a new substance, and can be separated with ethyl acetate: n-hexane of 93:7 by a high performance thin layer chromatography(HPTLC), has a selective proliferation-inhibiting activity, and can be used for the treatments of malignant cancers of uterine cervical cancer, vulvar cancer, penile cancer, laryngeal cancer and carcinoma cutaneum. The antitumor substance is obtained by grinding vegetables, adding distilled water, centrifuging the mixture, dialyzing the supernatant, concentrating the dialyzed liquid, extracting the concentration product with ethyl acetate in an acidic state, and developing and separating the extract with a high performance thin layer plate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a vegetable-derived antitumor substance having a selective growth inhibitory activity against tumor cells and a method for producing the same.

[0002] Study on the anti-tumor substance of the background art vegetables is done many, also, a number of reports on this have been made (VLSp
arnins et al ., Carcinogenesis , 9 , 131 (1988); MJWa
rgovich et al., Cancer Research, 48,6872 (1988);
M. Murakoshi et al ., Cancer Research , 52 , 6583-6587 (1
992);. GDstoner et al, Cancer Researc h, 51, 2063 (1
Natl . Acad . Sci . USA , Y. Zhang et al ., Proc . Natl .
89 , 2394 (1992); Y. Zhang et al ., Proc . Natl . Acad .
Sci . USA , 91 , 3147 (1994)). The types are diverse and various organic compounds have been reported.

Recently, maize ( Zea Mays L.) has been used as a low-molecular-weight organic compound in vegetables that is directly cytotoxic to tumor cells in a test method using cultured cells in vitro.
13-Hydroxy-10-oxo-trans-11-
Octadecenoic acid (Abstracts of the Japanese Society for Agricultural Chemistry 1993)
6- Methylsulfinylhexylisothiocyanate from Wastreme ( Etrema wasabi Maxim.) (Journal of the Japan Society for Food Engineering, 43 , 1092-10997 (1996))
Etc. have been reported.

[0004] An antitumor fraction of pure corn vinegar, which has been confirmed to have a selective growth inhibitory activity on tumor cells using SV40-transformed cells (Abstracts of the Japanese Society for Agricultural Chemistry, 1989). However, no identification of an antitumor substance derived from pure corn vinegar has been made.

[0005]

SUMMARY OF THE INVENTION The present inventors have now proposed radish ( Raphan
us sativus L. var.), Japanese radish ( Raphanus )
sativus L. var.), garlic ( Allium sativum )
L.), garlic ( Allium sativum L.) sprouts, Rakkyo ( Allium Bakeri ), ginger ( Zingiber offi )
In the organic solvent extract of C. cinale ), a substance having high antitumor activity and selective growth inhibitory activity against tumor cells was found. The present invention is based on this finding.

Accordingly, an object of the present invention is to provide a vegetable-derived antitumor substance, a pharmaceutical composition containing the antitumor substance, and a method for producing the antitumor substance.

The antitumor substance according to the present invention is a vegetable-derived substance having the following properties (1) to (4): (1) It has a molecular weight of 3,500 or less as measured by a dialysis membrane. 2) extracted by an organic solvent, (3) a neutral substance, (4) high performance thin layer chromatography (HP
TLC) can be separated by developing with ethyl acetate: n-hexane = 93: 7.

The antitumor substance according to the present invention is considered to be a neutral low molecular organic compound and has excellent antitumor activity.

[0009]

DETAILED DESCRIPTION OF THE INVENTION Anti-tumor substances The anti-tumor substances according to the invention originate from vegetables. In the present invention, "vegetables" refers to the family Nymphalidaceae, Nymphalidae, Umbrella-flowered Apiaceae, Coleoptera Akazaeaceae, Coleoptera brassicaceae,
Roses, legumes, tubular flowers, convolvulaceae and solanaceae, sea urchins, cucurbits, asters, asteraceae, spikelets, taros, sweet potatoes, lily lilies and yams, and ginger ginger. Means plant. Examples of vegetables having a fraction exhibiting good antitumor activity include, for example, cruciferous plants (for example, Raphanus radish ( Raphanus radish)
sativus L. var.), a lily plant (for example, garlic ( Allium sativum L.)), and a ginger plant (for example, ginger ( Zingiber of ficinale )).

[0010] The antitumor substance according to the present invention is extracted with an organic solvent. Here, the “organic solvent” is used in a sense including ethyl acetate, n-hexane, ether, chloroform, and the like.

The antitumor substance according to the present invention is a neutral substance. Here, "neutral substance" refers to a substance that is not extracted into an acidic solution having a pH of 2 to 4 and an alkaline solution having a pH of 9 to 12 (see Examples described later).

The antitumor substance according to the present invention can be separated by high performance thin layer chromatography (HPTLC) by developing with ethyl acetate: n-hexane = 93: 7.
Rf values by HPTLC of the antitumor substances derived from radish, garlic, and ginger are as follows. Red radish: 0.15; Garlic: 0.57 and 0.95; Ginger: 0.83.

[0013] The antitumor activity of the present invention is determined by using mouse normal cells (for example, fetal fibroblasts) as described below.
It can be confirmed by using a cell (tumor cell) transformed with a tumor virus (for example, Papovaviridae) and measuring the selective growth inhibitory activity against the tumor cell. Here, “has an antitumor activity” means that it exhibits a growth inhibitory activity on tumor cells and shows a growth inhibitory activity on tumor cells more than normal cells.

Substances derived from radish, garlic, and ginger show high antitumor activity and have a selectivity of 2.0 or more in Swiss cells. I of normal cells
The value obtained by dividing the C ₅₀ value by the IC ₅₀ value of the tumor cell was defined as “selection ratio”, which was used as an index of selectivity.

Method for producing antitumor substance The antitumor substance according to the present invention is basically obtained by subjecting a commercially available vegetable to aqueous solvent extraction and dialysis to obtain a molecular weight of 3,50.
The target product can be obtained by collecting fractions of 0 or less, further extracting with an organic solvent (for example, ethyl acetate), and removing the acidic extraction fraction and the basic extraction fraction. Furthermore, a step of developing and separating with ethyl acetate: n-hexane = 93: 7 by high performance thin layer chromatography (HPTLC) may be performed.

The vegetables to be extracted are preferably finely crushed as an extraction material in consideration of extraction efficiency.

The method for producing an antitumor substance according to the present invention can be carried out as follows. First, raw or frozen vegetables (thaw if frozen) are crushed with a homogenizer or the like, and an appropriate amount (preferably about the same amount) of an aqueous solvent is added thereto. Stir and extract under cooling. In this case, the “aqueous solvent” is a solvent mainly composed of water, and refers to water or a buffer solution (preferably about pH 7) containing a suitable inorganic acid salt or organic acid salt such as a PBS solution. The extract is centrifuged at a low temperature (preferably about 4 ° C.) (usually 10,000 to 40,
000 × g for 10 to 45 minutes) to obtain a crude extract. Using a dialysis membrane having a cut-off molecular weight of 3,500, the crude extract is exchanged with water at low temperature (preferably about 1 to 10 ° C.) for an appropriate time, preferably about 24 hours, while exchanging the external solution several times. Dialyze. The outer dialysis solution is concentrated under reduced pressure at room temperature (preferably 35 ° C. or lower) by, for example, a vacuum pump and a rotary evaporator to obtain a low molecular fraction (FIG. 1).
(A)).

The low molecular fraction is acidified with hydrochloric acid (eg, pH
2-4, preferably about pH 3), transferred to a separatory funnel and extracted with ethyl acetate to obtain an ethyl acetate phase while preventing the basic substance contained in the aqueous phase from being mixed. The strongly acidic fraction and the weakly acidic fraction are removed from the ethyl acetate phase with an alkaline aqueous solution (preferably, an aqueous solution of sodium hydrogen carbonate of about pH 9 and an aqueous solution of sodium hydroxide of about pH 12),
Ethyl acetate is distilled off to obtain the desired product (neutral fraction) (see FIG. 2).

Further, these neutral fractions were subjected to high performance thin layer chromatography (HPTLC), and a suitable organic solvent (preferably ethyl acetate: n-hexane = 93: 7) was used.
At a suitable distance (preferably about 6 cm), and irradiate with ultraviolet light having a wavelength of 2536 angstroms or 3650 angstroms to detect the Rf value (the development distance of the organic solvent used for the development based on the development distance of the target substance). The Rf value is determined, the detected substance having the Rf value is scraped off, extracted with a suitable organic solvent (preferably methanol), and the organic solvent is distilled off to obtain the desired product (FIG. 1). (B)).

The purity of these target substances can be further increased by a purification means such as column chromatography, if necessary.

The target product thus obtained has the above-mentioned physicochemical properties (1) to (4) and has antitumor activity (see Examples below).

The antitumor activity can be measured by a commonly used method, for example, a method using a mouse transplanted with a tumor (Ki
kuo Nomoto, Chikao Yoshikumi, Kenichi Matunaga, Takay
oshiFujii and Kenji Takeya “Restoration of Antibo
dy-forming Capacities byPS-K in Tumor-bearing Mic
e " Gann 66 (1975) 365-374). However, by measuring cytostatic activity in vitro using animal cells transformed with tumor viruses, the equipment required for research can be This is a significant advantage compared to the in vivo method in terms of cost, time, etc. A preferred example of a method for measuring cytostatic activity in vitro is described in Example 2.

When the antitumor agent according to the present invention is used as an antitumor agent as described below, usually, a low molecular fraction is recovered from an aqueous extract of vegetables by dialysis and extracted with ethyl acetate as an active substance, or Further, those obtained by high performance thin layer chromatography (HPTLC) are used,
An aqueous extract of vegetables was directly extracted with ethyl acetate, or the extract was subjected to high performance thin layer chromatography (HPTLC) or thin layer chromatography (TL).
Those obtained by C) can also be used (see Examples below). The latter is advantageous from the viewpoint of mass purification.

[0024] As described above antitumor agents, antitumor agents according to the invention have a selective growth inhibitory activity against tumor cells. Therefore, according to another aspect of the present invention, there is provided a pharmaceutical composition comprising the antitumor substance.

The pharmaceutical composition according to the present invention can be used for the treatment of malignant tumors (for example, cervical cancer, vulvar cancer, penis cancer, laryngeal cancer, skin cancer, etc.).

The therapeutic agent according to the present invention can also be administered orally or parenterally (eg, intramuscular, intravenous, subcutaneous, rectal, transdermal, nasal, sublingual, etc.) It is used in humans and non-human animals in various dosage forms suitable for oral or parenteral administration as medicaments. It is considered that a method of directly reaching a diseased part as a drug (for example, a locally soluble tablet, application, injection, or the like) is effective.

The therapeutic agent according to the present invention takes into account, for example, the stability of the protein such as tablets, capsules, granules, powders, pills, fine granules, and troches, and the drug delivery route, depending on its use. The preparation can be prepared in any formulation form such as oral preparations, injection preparations such as intravenous injection and intramuscular injection, rectal administration preparations, oily suppositories, water-soluble suppositories and the like. These various preparations, commonly used excipients, for example, fillers, binders, wetting agents, disintegrants, surfactants, lubricants,
It can be produced by a conventional method using a dispersant, a buffer, a preservative, a solubilizing agent, a preservative, a flavoring agent, a soothing agent, a stabilizer and the like.

The dosage for various treatments can be appropriately determined in consideration of the usage, the age, sex, degree of symptoms, etc. of the patient.

[0029]

The present invention will be described in detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

Example 1: Extraction of test substance 60 kinds of commercially available 40 kinds of vegetables (see Table 1)
Was crushed with a homogenizer, an equal amount of distilled water was added thereto, and the mixture was stirred for several minutes. The crushed liquid was centrifuged at a low temperature (4 ° C.) (36,000 × g, 15 minutes) to obtain a crude extract (first screening sample).

With respect to this crude extract, the molecular weight cutoff was 3,5.
00 dialysis membrane (Spectra / Pore 3, SPECTRUM
For 8 hours at a low temperature (4 ° C.)
Dialysis was performed. The outer dialysate was concentrated under reduced pressure at room temperature (30 ° C.) using a vacuum pump and a rotary evaporator to obtain a low molecular fraction (second screening sample).

The low molecular fraction was made acidic (pH 3) with hydrochloric acid, transferred to a separating funnel and extracted with ethyl acetate, to obtain an ethyl acetate phase (acidic fraction, neutral fraction). The aqueous phase obtained at this time was made alkaline (pH: 10%) with a 10% aqueous sodium hydroxide solution.
12) and extracted with ethyl acetate to obtain an aqueous phase (amphoteric fraction) and an ethyl acetate phase (basic fraction). On the other hand, from the ethyl acetate phase (acidic fraction, neutral fraction), an alkaline aqueous solution (5%
The strongly acidic fraction was separated with an aqueous sodium hydrogen carbonate solution (pH 9), and further a 5% aqueous sodium hydroxide solution (pH 1) was added.
The weakly acidic fraction was separated in 2), and ethyl acetate was distilled off to obtain a neutral fraction. Fractionation by solvent extraction is as shown in FIG.

Japanese radish, garlic, ginger,
The neutral fraction of garlic sprouts, radish, and rakkyo is dissolved in ethanol, and the mixture is subjected to a high-performance thin-layer plate (HPTLC plate silica gel 60 F ₂₅₄ , size 10 cm × 10
cm, manufactured by Merck Co., Ltd.), developed with an organic solvent of ethyl acetate: n-hexane = 93: 7 for 6 cm, air-dried, and then irradiated with ultraviolet light of 2536 Å or 3650 Å. Irradiation and detection were performed to determine the Rf value.
The substance exhibiting the Rf value was scraped off, extracted with methanol, and the methanol was distilled off to obtain an HPTLC separated sample. The result of HPTLC was as shown in FIG.

Similarly, those obtained by directly extracting an aqueous extract of vegetables with ethyl acetate without dialysis, or obtained from the extract by high-performance thin-layer chromatography (HPTLC) or thin-layer chromatography (TLC) are also used. A test sample was used.

Example 2: Anti-resistance of organic solvent extract derived from vegetables
Tumor activity measurement mouse fetal fibroblast 3T3 (3T3-Swissa
lbino, derived from Swiss albino mouse, RIKEN Genebank (within RIKEN), BALB / 3T3 (BAL
B / 3T3 clone A31, derived from BALB / c mouse, RIKEN GeneBank), and 3T3-SV40 (RIKEN GeneBank) obtained by transforming them with papovavirus SV40, and BALB / 3T3-SV40 (RIKEN GeneBank) as 10% offspring. The seeds were bred in a bovine serum-containing medium (Dulbecco's modified Eagle medium) at 2 × 10 ⁴ cells / ml, and cultured for 24 hours at 37 ° C. under 5% CO ₂ . Here, the neutral fraction of each vegetable prepared in Example 1 and H
A 10% ethanol aqueous solution of the PTLC separated sample was added, and the cells were further cultured for 48 hours, and the number of cells was measured by the MTT method.

The MTT method was performed as follows. First,
Four hours before measurement, cells were treated with MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromine) in phosphate-buffered saline (PBS solution: 137 mM NaCl, 2. 7 mM KCl, 8.
1 mM sodium phosphate-hydrogen phosphate and 1.5 mM potassium dihydrogen phosphate (pH 7.3 to 7.6) were added, and the cells were further cultured for 4 hours. Next, after removing the culture supernatant, an aqueous solution of hydrochloric acid (0.04N) isopropanol-3% SDS was added to dissolve formazan generated by the action of mitochondria of living cells, and then the absorbance at 595 nm (control 655 nm) of the solution was measured. (Tim Mosmann “Rapid
Colorimetric Assay for Cellular Growth and Surviva
l: Application to Proliferation and Cytotoxicity A
ssays ” Journal of Immunological M ethods 65 (1983) 5
5-63; Lora M. Green, Jeanne L. Reade and Carl F.Ware
“Rapid Colormetric Assay for Cell Viability: Appli
cation to the Quantitation of Cytotoxic and Growth
Inhibitory Limphokines ” Journal of Immunological
Methods 70 (1984) 257-268).

The sample concentration of the test substance was measured at several points, and the concentration of the sample showing 50% growth inhibition relative to the number of control cells was expressed as IC ₅₀ value. Furthermore, the value (selectivity) obtained by dividing the IC ₅₀ value of normal cells by the IC ₅₀ value of tumor cells was used as an index for evaluating antitumor activity. The sample concentration was expressed as a dry weight concentration.

The results of the antitumor test were as shown in the following table.

[0039]

[Table 1] Antitumor activity was observed in 15 of the 40 vegetables. High selectivity was observed mainly in cruciferous, lily and ginger plants.

[0040]

[Table 2] The high molecular fraction of molecular weight of radish, corn, asparagus, and garlic (molecular weight of 3,500 or more) and the low molecular weight fraction of molecular weight of broccoli, radish, radish, corn, garlic, garlic sprouts, rakkyo, and ginger (molecular weight of 3,500 In the following, high selectivity was observed.

[0041]

[Table 3] Neutral fractions derived from radish, garlic, and ginger exhibited a growth inhibitory activity against tumor cells and a selectivity ratio exceeding 1.0 in any of the Swiss albino strain and the BALB / c strain, and exhibited antitumor activity.

[0042]

[Table 4] The HPTLC-isolated sample of the neutral fraction from the Japanese radish, garlic and ginger showed a high selectivity of over 2.0 in the Swiss albino system.

In addition, those obtained by directly extracting the aqueous extract of vegetables with ethyl acetate without dialysis, or those obtained from the extract by high performance thin layer chromatography (HPTLC) or thin layer chromatography (TLC) are also available. Showed equivalent antitumor activity (data not shown).

[Brief description of the drawings]

Fig. 1: Neutral fraction sample (a) from vegetables and HPTL
It is a figure which showed the outline of the preparation method of C isolation | separation sample (b), and the measurement method (c) of antitumor activity.

FIG. 2 is a view showing a step of solvent extraction from a low molecular fraction (aqueous solution).

FIG. 3 shows a neutral fraction extracted from vegetables with ethyl acetate by HPT
It is the figure which showed the result which applied LC. The black portions indicate spots having antitumor activity in Swiss cells. Shaded areas indicate spots that may have anti-tumor activity in Swiss cells. The numbers are the selectivity. A used ethyl acetate-n-hexane (93: 7) as a developing solvent, and B used benzene-methanol (7: 3) as a developing solvent.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Kazuaki Kido 410 Akanuma-do, Kasukabe-shi, Saitama Prefecture Momoya Laboratory Co., Ltd. (72) Inventor Yoshiko Aoki 410 Akanuma-do, Kasukabe-shi, Saitama Momo Inside the ya laboratory

Claims (14)

[Claims] 1. A vegetable-derived antitumor substance having the following properties (1) to (4): (1) having a molecular weight of 3,500 or less as measured by a dialysis membrane, and (2) being extracted with an organic solvent. (3) Neutral substances, (4) high performance thin layer chromatography (HP
TLC) can be separated by developing with ethyl acetate: n-hexane = 93: 7. 2. The antitumor substance according to claim 1, wherein the vegetable is a plant belonging to a family selected from the group consisting of Brassicaceae, Lily family, and Ginger family. 3. Vegetable is radish ( Raphanus sa)
tivus L. var.), garlic ( Alli um sativum L.)
2. The antitumor substance according to claim 1, which is selected from the group consisting of: ginger ( Zingiber officinale ). 4. An organic solvent comprising ethyl acetate, n-hexane,
The antitumor substance according to claim 1, which is selected from the group consisting of ether and chloroform. 5. An acidic solution of pH 2-4 and pH 9-12.
The antitumor substance according to claim 1, which is not extracted into an alkaline solution of: 6. The method according to claim 1, wherein the tumor cells are mouse fetal fibroblasts transformed with a DNA oncovirus.
2. The antitumor substance according to 1. 7. An antitumor substance derived from Japanese radish ( Raphanus sativus L. var.) Having the following properties (1) to (5): (1) a molecular weight of 3,500 or less as measured by a dialysis membrane; 2) extracted by an organic solvent, (3) a neutral substance, (4) high performance thin layer chromatography (HP
Ethyl acetate: n-hexane = 93: 7 by TLC)
The Rf value by HPTLC of (5) and (4), which can be developed and separated, is 0.15. 8. An antitumor substance derived from garlic ( Allium sativum L.) having the following properties (1) to (5): (1) a molecular weight of 3,500 or less as measured by a dialysis membrane; (2) (3) neutral substance, (4) high performance thin layer chromatography (HP
Ethyl acetate: n-hexane = 93: 7 by TLC)
Rf values by HPTLC of (5) and (4) are 0.57 and 0.95, which can be separated and developed. 9. An antitumor substance derived from ginger ( Zingiber officinale ) having the following properties (1) to (5): (1) having a molecular weight of 3,500 or less as measured by a dialysis membrane, and (2) an organic solvent. (3) neutral substance, (4) high performance thin layer chromatography (HP
Ethyl acetate: n-hexane = 93: 7 by TLC)
The Rf value by HPTLC of (5) and (4), which can be developed and separated, is 0.83. 10. The selection ratio in Swiss-type cells is 2.
The antitumor substance according to any one of claims 7 to 9, which is 0 or more. A pharmaceutical composition comprising the antitumor substance according to any one of claims 1 to 10. 12. The method according to claim 1, which is used for treating a malignant tumor.
2. The pharmaceutical composition according to 1. 13. The method for producing an antitumor substance according to claim 1, comprising the following steps. (1) Vegetables are subjected to aqueous solvent extraction, (2) fractions having a molecular weight of 3,500 or less are collected from the aqueous extract by a dialysis membrane, (3) extracted with an organic solvent, and (4) acidic The extraction fraction and the basic extraction fraction are removed. 14. High performance thin layer chromatography (HPT)
14. The production method according to claim 13, further comprising a step of developing and separating with ethyl acetate: n-hexane = 93: 7 by LC).